Complicating 5-8% of all pregnancies, preeclampsia (PE) is one of the three main causes of premature birth. The most notable characteristics of PE are hypertension, edema and excess protein in the urine (proteinuria) after the 20th week of pregnancy. Consequences for the fetus can be grave, ranging from small-for-gestational-age infancy to hypoxia-induced neurologic injury (e.g., cerebral palsy) to death. Maternal complications include renal failure, HELLP syndrome (Hemolysis, Elevated Liver enzymes, and Low Platelets), seizures, stroke, and death. PE and related hypertensive disorders are conservatively estimated to cause 76,000 maternal and 500,000 infant deaths globally each year. (See preeclampsia [dot] org.) In the United States, PE is responsible for 100,000 premature births and 10,500 infant deaths each year at a cost of roughly seven billion dollars (three billion dollars for maternal disabilities and four billion dollars related to infant morbidity) every year to the health care system. Across the globe, PE and subsequent eclampsia are major contributors to maternal, fetal and neonatal morbidity and mortality. Thus, PE represents a highly significant unmet public health need.
Although the root causes of PE have yet to be fully understood, it is now well established that the maternal signs and symptoms of hypertension, edema and proteinuria are caused by an excess of anti-angiogenic proteins in the mother's bloodstream. Chief among these are soluble fins-like tyrosine kinase 1 (sFLT1s) proteins. sFLT1s are truncated forms of the membrane-bound vascular endothelial growth factor (VEGF) receptor FLT1 (also known as VEGFR1). They normally function to buffer VEGF signaling. However, when sFLT1s are abnormally high in the mother's circulatory system, they can interfere with her body's own ability to respond to VEGF. Among other functions, VEGF is required for maintenance of the hepatic sinusoidal vasculature and other fenestrated vascular beds in the body (Kamba, T. et al. VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature. American journal of physiology. Heart and circulatory physiology 290, H560-576 (2006)). Breakdown of these structures impairs maternal kidney function, leading to hypertension, proteinuria and cerebral edema which are classic features of PE and eclampsia (Young, B. C., Levine, R. J. & Karumanchi, S. A. Pathogenesis of preeclampsia. Annual review of pathology 5, 173-192 (2010); Eremina, V. et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. The Journal of clinical investigation 111, 707-716 (2003); Eremina, V. et al. VEGF inhibition and renal thrombotic microangiopathy. The New England journal of medicine 358, 1129-1136 (2008)).
Pilot studies using an extracorporeal device to remove sFLT1 from the bloodstream of severely preeclamptic women has demonstrated that lowering sFLT1 protein by just 30-40% in the maternal plasma can prolong PE pregnancies by 2 weeks without adverse consequences to the baby (Thadhani, R. et al. Pilot study of extracorporeal removal of soluble fins-like tyrosine kinase 1 in preeclampsia. Circulation 124, 940-950 (2011)). Moreover, animal studies support the hypothesis that targeting sFLT1 in PE may also lower the risk of neonatal respiratory problems and bronchopulmonary dysplasia, major complications of prematurity (Tang, J. R., Karumanchi, S. A., Seedorf, G., Markham, N. & Abman, S. H. Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia. American journal of physiology. Lung cellular and molecular physiology 302, L36-46 (2012)). Yet, while apheresis (blood washing) is highly promising, it is unlikely to be applicable to all patients in all situations. Especially in low resource settings, a more cost effective approach with lower medical and general infrastructure requirements is desperately needed. RNA silencing via RNAi is one such approach.
A broad range of human diseases, including cancer, infection and neurodegeneration, can be treated via the silencing of specific genes using small oligonucleotides. ONTs (OligoNucleotide Therapeutics) are a new class of drugs, distinguished by targeting RNA or DNA directly, thus interfering with a disease-causing gene at its root, before it can produce the protein responsible for the disease phenotype. Advantages of ONTs over conventional drugs include ease of drug design based solely on base-pairing rules, an ability to access targets previously considered “undruggable” and their promise of unprecedented specificity, potency, and duration of effect. In addition, pharmacokinetics, pharmacodynamics and safety of ONTs is mostly defined by chemical modifications/formulation and is very similar between compound targeting different genes, enabling multi-gene silencing and simple development drugs targeting the same tissue (Videira, M., Arranja, A., Rafael, D. & Gaspar, R. Preclinical development of siRNA therapeutics: towards the match between fundamental science and engineered systems. Nanomedicine: nanotechnology, biology, and medicine 10, 689-702 (2014); H. Youths et al. in A Comprehensive Guide to Toxicology in Preclinical Drug Development. (ed. A. S. Faqi) 647-664 (Academic Press, 2013)). Significant effort in the last decade resulted in development of several types of both chemically-modified and formulated oligonucleotides with clear clinical efficacy (Whitehead, K. A., Langer, R. & Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nature reviews. Drug discovery 8, 129-138 (2009)). Thus, ONTs represent a new and potentially transformative therapeutic paradigm. Nonetheless, their clinical utility has been hampered by limited tissue distribution. Systemic administration has been generally limited to liver hepatocytes to date, with other tissues requiring local administration (de Fougerolles, A., Vornlocher, H. P., Maraganore, J. & Lieberman, J. Interfering with disease: a progress report on siRNA-based therapeutics. Nature reviews. Drug discovery 6, 443-453 (2007)).
One class of ONTs is siRNAs, small double-stranded oligonucleotides consisting of passenger (sense) and guide (antisense) strands. Upon cellular uptake, the guide strand is loaded into an RNA Induced Silencing Complex (RISC) capable of cleaving its complementary target RNA. The numbers of loaded RISCs per cell sufficient to induce efficient and long-term gene silencing or RNA interference (RNAi) are estimated at approximately 25-100 in vitro (Stalder, L. et al. The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing. The EMBO journal 32, 1115-1127 (2013)) and approximately 400 in vivo (Pei, Y. et al. Quantitative evaluation of siRNA delivery in vivo. Rna 16, 2553-2563 (2010)). Typically, 10-100 ng/gram of oligonucleotide delivered to a targeted tissue (Overhoff, M., Wunsche, W. & Sczakiel, G. Quantitative detection of siRNA and single-stranded oligonucleotides: relationship between uptake and biological activity of siRNA. Nucleic acids research 32, e170 (2004)) is adequate to generate a sufficient number of active RISC complexes and induce silencing. Loaded RISCs have weeks long stability, resulting in prolonged gene silencing (3-6 weeks) from a single administration (Whitehead, K. A., Langer, R. & Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nature reviews. Drug discovery 8, 129-138 (2009)).